Green Fluorescent Protein Chromophore Research Articles

NanoLuc Luciferase as a Fluorogen-Activating Protein for GFP Chromophore Based Fluorogens.

In this work, we showed that the well-known NanoLuc luciferase can act as a fluorogen activating protein for various arylidene-imidazolones structurally similar to the Kaede protein chromophore. We showed that such compounds can be used as fluorescent sensors for this protein and can also be used in pairs with it in fluorescent microscopy as a genetically encoded tag.

Quantum mechanics/fluctuating charge calculations for absorption spectra of aqueous green fluorescent protein chromophore

AbstractWe applied the quantum mechanics/fluctuating charges (QM/FQ) method to simulate the absorption spectra of three protonated forms of p‐hydroxybenzylidene imidazolone (p‐HBDI) in water. Configurations of solute‐solvent were generated from molecular dynamics (MD) simulation within the context of a hybrid scheme in which solute and solvents were treated at QM and molecular mechanics, respectively. Nonperiodic spherical boundary condition was used. To examine the influence on simulated spectra coming from the geometrical relaxation of solute, the MD simulations were carried out for two cases: frozen and flexible molecular geometry of solute. As a result of our calculations, the simulations using the flexible solute's structure delivered vertical excitation energies closer to experimental data than those of a frozen solute, especially for the neutral case. For spectral broadening, changing from fixed geometry of solute to a flexible one led to a significant increase for both neutral and anionic forms of p‐HBDI, but no effect happened for the cationic one.

Multiple Photoisomerization Pathways of the Green Fluorescent Protein Chromophore in a Reversibly Photoswitchable Fluorescent Protein: Insights from Quantum Mechanics/Molecular Mechanics Simulations

Herein, we have employed a combined CASPT2//CASSCF approach within the quantum mechanics/molecular mechanics (QM/MM) framework to explore the early time photoisomerization of rsEGFP2 starting from its two OFF trans states, i.e., Trans1 and Trans2. The results show similar vertical excitation energies to the S1 state in their Franck-Condon regions. Considering the clockwise and counterclockwise rotations of the C11-C9 bond, four pairs of the S1 excited-state minima and low-lying S1/S0 conical intersections were optimized, based on which we determined four S1 photoisomerization paths that are essentially barrierless to the relevant S1/S0 conical intersections leading to efficient excited-state deactivation to the S0 state. Most importantly, our work first identified multiple photoisomerization and excited-state decay paths, which must be seriously considered in the future. This work not only sheds significant light on the primary trans-cis photoisomerization of rsEGFP2 but also aids in the understanding of the microscopic mechanism of GFP-like RSFPs and the design of novel GFP-like fluorescent proteins.

Excited State Intramolecular Proton Transfer Dynamics of Derivatives of the Green Fluorescent Protein Chromophore

Excited state intramolecular proton transfer (ESIPT) dynamics of the o-hydroxy analogs of the green fluorescent protein (GFP) chromophore have been investigated by time-resolved spectroscopies and theoretical calculations. These molecules comprise an excellent system to investigate the effect of electronic properties on the energetics and dynamics of ESIPT and to realize applications in photonics. Time-resolved fluorescence with high enough resolution was employed to record the dynamics and the nuclear wave packets in the excited product state exclusively in conjunction with quantum chemical methods. The ESIPT are ultrafast occurring in 30 fs for the compounds employed in this work. Although the ESIPT rates are not affected by the electronic properties of the substituents suggesting barrierless reaction, the energetics, their structures, subsequent dynamics following ESIPT, and possibly the product species are distinct. The results attest that fine tuning of the electronic properties of the compounds may modify the molecular dynamics of ESIPT and subsequent structural relaxation to achieve brighter emitters with broad tuning capabilities.

Carbon-deuterium bonds as reporters of electric fields in solvent and protein environments.

Use of vibrational probes to measure electric fields inside proteins has illuminated the role of electrostatics in biological applications ranging from binding interactions to enzyme catalysis. While prior work focused on strongly absorbing carbonyl and nitrile probes, more recent work has begun exploring weakly absorbing carbon-hydrogen bonds, beginning with measurements of electric fields inside oxidoreductases using the vibrational Stark effect (VSE). Originally, an alcohol dehydrogenase inhibitor containing a deuterated aldehyde was used to measure the field in two directions—on carbonyl and carbon-deuterium (C-D) bonds—in the enzyme active site. Now, several more distinct cases demonstrate how C-D probes map electric fields to vibrational frequency shifts, with new implications for our understanding of the VSE. To probe the field along a different dimension in oxidoreductases, we consider singly deuterated NAD(P)H which reports fields along the hydride transfer axis and whose frequency detection is facilitated by advancements in quantum cascade laser (QCL) technology for infrared spectroscopy. Unusually, the solvatochromic shift for this C-D probe appears opposite to that observed for carbonyls and nitriles; that is, the probe blueshifts in more polar solvents. In another example, the chromophore of green fluorescent protein (GFP) with a deuterated bridge carbon demonstrates similarly unusual frequency shifts in solvent and protein environments, with stronger fields parallel to the C-D dipole causing blueshifts. Observed Stark tuning rates for these molecules are recapitulated by in silico Stark calculations at the B3LYP or ωB97X-D/6-31+G(d) level. Stark tuning rate comparison with other C-D containing molecules including deuterated aldehydes and chloroform-d indicates a large sensitivity of the Stark tuning rate to molecular structure. We demonstrate how properly calibrated C-D probes can be effective reporters of electric fields in applications such as enzyme-catalyzed hydride transfer, biological photophysics and beyond.

Efficient All-Electron Time-Dependent Density Functional Theory Calculations Using an Enriched Finite Element Basis.

We present an efficient and systematically convergent approach to all-electron real-time time-dependent density functional theory (TDDFT) calculations using a mixed basis, termed as enriched finite element (EFE) basis. The EFE basis augments the classical finite element basis (CFE) with a compactly supported numerical atom-centered basis, obtained from atomic ground-state DFT calculations. Particularly, we orthogonalize the enrichment functions with respect to the classical finite element basis to ensure good conditioning of the resultant basis. We employ the second-order Magnus propagator in conjunction with an adaptive Krylov subspace method for efficient time evolution of the Kohn-Sham orbitals. We rely on a priori error estimates to guide our choice of an adaptive finite element mesh as well as the time step to be used in the TDDFT calculations. We observe close to optimal rates of convergence of the dipole moment with respect to spatial and temporal discretizations. Notably, we attain a 50-100 times speedup for the EFE basis over the CFE basis. We also demonstrate the efficacy of the EFE basis for both linear and nonlinear responses by studying the absorption spectra in sodium clusters, the linear to nonlinear response transition in the green fluorescence protein chromophore, and the higher harmonic generation in the magnesium dimer. Lastly, we attain good parallel scalability of our numerical implementation of the EFE basis for up to ∼1000 processors, using a benchmark system of a 50-atom sodium nanocluster.

Alkylated green fluorescent protein chromophores: dynamics in the gas phase and in aqueous solution.

Fluorescent labelling of macromolecular samples, including using the green fluorescent protein (GFP), has revolutionised the field of bioimaging. The ongoing development of fluorescent proteins require a detailed understanding of the photophysics of the biochromophore, and how chemical derivatisation influences the excited state dynamics. Here, we investigate the photophysical properties associated with the S1 state of three alkylated derivatives of the chromophore in GFP, in the gas phase using time-resolved photoelectron imaging, and in water using femtosecond fluorescence upconversion. The gas-phase lifetimes (1.6-10 ps), which are associated with the intrinsic (environment independent) dynamics, are substantially longer than the lifetimes in water (0.06-3 ps), attributed to stabilisation of both twisted intermediate structures and conical intersection seams in the condensed phase. In the gas phase, alkylation on the 3 and 5 positions of the phenyl ring slows the dynamics due to inertial effects, while a 'pre-twist' of the methine bridge through alkylation on the 2 and 6 positions significantly shortens the excited state lifetimes. Formation of a minor, long-lived (≫ 40 ps) excited state population in the gas phase is attributed to intersystem crossing to a triplet state, accessed because of a T1/S1 degeneracy in the so-called P-trap potential energy minimum associated with torsion of the single-bond in the bridging unit connecting to the phenoxide ring. A small amount of intersystem crossing is supported through TD-DFT molecular dynamics trajectories and MS-CASPT2 calculations. No such intersystem crossing occurs in water at T = 300 K or in ethanol at T ≈ 77 K, due to a significantly altered potential energy surface and P-trap geometry.

Protomers of the green and cyan fluorescent protein chromophores investigated using action spectroscopy.

The photophysics of biochromophore ions often depends on the isomeric or protomeric distribution, yet this distribution, and the individual isomer contributions to an action spectrum, can be difficult to quantify. Here, we use two separate photodissociation action spectroscopy instruments to record electronic spectra for protonated forms of the green (pHBDI+) and cyan (Cyan+) fluorescent protein chromophores. One instrument allows for cryogenic (T = 40 ± 10 K) cooling of the ions, while the other offers the ability to perform protomer-selective photodissociation spectroscopy. We show that both chromophores are generated as two protomers when using electrospray ionisation, and that the protomers have partially overlapping absorption profiles associated with the S1 ← S0 transition. The action spectra for both species span the 340-460 nm range, although the spectral onset for the pHBDI+ protomer with the proton residing on the carbonyl oxygen is red-shifted by ≈40 nm relative to the lower-energy imine protomer. Similarly, the imine and carbonyl protomers are the lowest energy forms of Cyan+, with the main band for the carbonyl protomer red-shifted by ≈60 nm relative to the lower-energy imine protomer. The present strategy for investigating protomers can be applied to a wide range of other biochromophore ions.

Photophysics of the red-form Kaede chromophore.

The green fluorescent protein (GFP) drove revolutionary progress in bioimaging. Photoconvertible fluorescent proteins (PCFPs) are an important branch of the FP family, of which Kaede is the prototype. Uniquely, PCFPs can be permanently switched from green to red emitting forms on UV irradiation, facilitating applications in site-specific photolabelling and protein tracking. Optimisation and exploitation of FPs requires understanding of the photophysical and photochemical behaviour of the chromophore. Accordingly, the principal GFP chromophore has been the subject of intense experimental and theoretical investigation. In contrast, the photophysics of the red emitting PCFP chromophore are largely unstudied. Here we present a detailed investigation of the excited-state properties of the Kaede chromophore in solution, utilising steady state measurements, ultrafast time-resolved electronic and vibrational spectroscopies, and electronic structure theory. Its excited state dynamics are very different to those of the parent GFP. Most remarkably, the PCFP chromophore has highly complex wavelength-dependent fluorescence decays and a mean lifetime an order of magnitude longer than the GFP chromophore. Transient electronic and vibrational spectroscopies suggest that these dynamics arise from a range of excited-state conformers that are spectrally and kinetically distinct but chemically similar. These conformers are populated directly by excitation of a broad thermal distribution of ground state structures about a single conformer, suggesting an excited-state potential surface with several minima. Temperature-dependence confirms the existence of barriers on the excited-state surface and reveals the radiationless decay mechanism to be internal conversion. These experimental observations are consistent with a model assuming a simple ground state potential energy surface accessing a complex excited state possessing multiple minima.

Excited-state dynamics and fluorescence lifetime of cryogenically cooled green fluorescent protein chromophore anions.

Time-resolved action spectroscopy together with a fs-pump probe scheme is used in an electrostatic ion-storage ring to address lifetimes of specific vibrational levels in electronically excited states. Here we specifically consider the excited-state lifetime of cryogenically cooled green fluorescent protein (GFP) chromophore anions which is systematically measured across the S0-S1 spectral region (450-482 nm). A long lifetime of 5.2 ± 0.3 ns is measured at the S0-S1 band origin. When exciting higher vibrational levels in S1, the lifetime changes dramatically. It decreases by more than two orders of magnitude in a narrow energy region ∼250 cm-1 (31 meV) above the 0-0 transition. This is attributed to the opening of internal conversion over an excited-state energy barrier. The applied experimental technique provides a new way to uncover even small energy barriers, which are crucial for excited-state dynamics.

Principles and applications of green fluorescent protein-based biosensors: a mini-review.

Green fluorescent proteins (GFPs) are useful and essential biomolecules that have revolutionized biosensor research. Researchers have extensively utilized GFPs for designing fluorescence biosensors due to their intrinsic fluorescence, high stability, and ability to undergo permutation or mutation. This review provides a concise summary of recent advancements in developing GFP-based biosensors. The construction approaches for GFP-based biosensors can be categorized into four types: (1) single GFP-based biosensors; (2) fluorescence resonance energy transfer-based biosensors; (3) GFP-based split biosensors; and (4) GFP chromophore analogy-based biosensors. We highlight the applications of these sensors in biomolecule detection and life sciences, while also sharing personal insights into the challenges associated with GFP-based biosensors and proposing future research directions.

Effects of hydrogen bonds on two-photon absorption of Green fluorescent protein chromophore analogue

Effects of hydrogen bonds on two-photon absorption (TPA) of a new donor-acceptor type green fluorescent protein chromophore analogue are investigated by employing a combined molecular dynamics and quantum chemistry method. The probable configurations of the chromophore in water are extracted from molecular dynamics simulation and the TP A properties of more than twenty hydrogen bond complexes are computed by quadratic response theory. Thereby, the structure and property relations are established. Three types of hydrogen bonds including O⋯H–O, N–H⋯O and N⋯H–O can be formed between the chromophore and water molecules. The O⋯H–O induces a little decrease of TPA cross section with a red-shifted wavelength. The N–H⋯O gives rise to a great enhancement of TP A at a longer wavelength, while the N⋯H–O decreases TP A significantly and makes the wavelength blue-shifted. The reasons for these effects are rationalized well by using a two-state model analysis. The related molecular orbitals are also plotted to visualize the charge transfer characters. In addition, the averaged TP A spectrum is obtained by calculating the probabilities of various hydrogen bond complexes. Our research could provide a good insight into the design of two-photon materials by making use of hydrogen bond networks.

7-membered-ring effect on fluorescence quantum yield: does metal-complexation-induced twisting-inhibition of an amino GFP chromophore derivative enhance fluorescence?

To investigate two aspects, namely, (1) the 7-membered-ring effect on fluorescence quantum yield and (2) whether metal-complexation-induced twisting-inhibition of an amino green fluorescent protein (GFP) chromophore derivative is bound to enhance fluorescence, a novel GFP-chromophore-based triamine ligand, (Z)-o-PABDI, is designed and synthesized. Before complexation with metal ions, the S1 excited state of (Z)-o-PABDI undergoes τ-torsion relaxation (Z/E photoisomerization) with a Z/E photoisomerization quantum yield of 0.28, forming both ground-state (Z)- and (E)-o-PABDI isomers. Since (E)-o-PABDI is less stable than (Z)-o-PABDI, it is thermo-isomerized back to (Z)-o-PABDI at room temperature in acetonitrile with a first-order rate constant of (1.366 ± 0.082) × 10-6 s-1. After complexation with a Zn2+ ion, (Z)-o-PABDI as a tridentate ligand forms a 1 : 1 complex with the Zn2+ ion in acetonitrile and in the solid state, resulting in complete inhibition of the φ-torsion and τ-torsion relaxations, which does not enhance fluorescence but causes fluorescence quenching. (Z)-o-PABDI also forms complexes with other first-row transition metal ions Mn2+, Fe3+, Co2+, Ni2+ and Cu2+, generating almost the same fluorescence quenching effect. By comparison with the 2/Zn2+ complex, in which a 6-membered ring of Zn2+-complexation enhances fluorescence significantly (a positive 6-membered-ring effect on fluorescence quantum yield), we find that the flexible 7-membered rings of the (Z)-o-PABDI/Mn+ complexes trigger their S1 excited states to relax through internal conversion at a rate much faster than fluorescence (a negative 7-membered-ring effect on fluorescence quantum yield), leading to fluorescence quenching regardless of the type of transition metal that complexes with (Z)-o-PABDI.

Locking the GFP Fluorophore to Enhance Its Emission Intensity.

The Green Fluorescent Protein (GFP) and its analogues have been widely used as fluorescent biomarkers in cell biology. Yet, the chromophore responsible for the fluorescence of the GFP is not emissive when isolated in solution, outside the protein environment. The most accepted explanation is that the quenching of the fluorescence results from the rotation of the aryl-alkene bond and from the Z/E isomerization. Over the years, many efforts have been performed to block these torsional rotations, mimicking the environment inside the protein β-barrel, to restore the emission intensity. Molecule rigidification through chemical modifications or complexation, or through crystallization, is one of the strategies used. This review presents an overview of the strategies developed to achieve highly emissive GFP chromophore by hindering the torsional rotations.

UV Photoelectron Spectroscopy of Aqueous Solutions.

ConspectusKnowledge of the electronic structure of an aqueous solution is a prerequisite to understanding its chemical and biological reactivity and its response to light. One of the most direct ways of determining electronic structure is to use photoelectron spectroscopy to measure electron binding energies. Initially, photoelectron spectroscopy was restricted to the gas or solid phases due to the requirement for high vacuum to minimize inelastic scattering of the emitted electrons. The introduction of liquid-jets and their combination with intense X-ray sources at synchrotrons in the late 1990s expanded the scope of photoelectron spectroscopy to include liquids. Liquid-jet photoelectron spectroscopy is now an active research field involving a growing number of research groups. A limitation of X-ray photoelectron spectroscopy of aqueous solutions is the requirement to use solutes with reasonably high concentrations in order to obtain photoelectron spectra with adequate signal-to-noise after subtracting the spectrum of water. This has excluded most studies of organic molecules, which tend to be only weakly soluble. A solution to this problem is to use resonance-enhanced photoelectron spectroscopy with ultraviolet (UV) light pulses (hν ≲ 6 eV). However, the development of UV liquid-jet photoelectron spectroscopy has been hampered by a lack of quantitative understanding of inelastic scattering of low kinetic energy electrons (≲5 eV) and the impact on spectral lineshapes and positions.In this Account, we describe the key steps involved in the measurement of UV photoelectron spectra of aqueous solutions: photoionization/detachment, electron transport of low kinetic energy electrons through the conduction band, transmission through the water-vacuum interface, and transport through the spectrometer. We also explain the steps we take to record accurate UV photoelectron spectra of liquids with excellent signal-to-noise. We then describe how we have combined Monte Carlo simulations of electron scattering and spectral inversion with molecular dynamics simulations of depth profiles of organic solutes in aqueous solution to develop an efficient and widely applicable method for retrieving true UV photoelectron spectra of aqueous solutions. The huge potential of our experimental and spectral retrieval methods is illustrated using three examples. The first is a measurement of the vertical detachment energy of the green fluorescent protein chromophore, a sparingly soluble organic anion whose electronic structure underpins its fluorescence and photooxidation properties. The second is a measurement of the vertical ionization energy of liquid water, which has been the subject of discussion since the first X-ray photoelectron spectroscopy measurement in 1997. The third is a UV photoelectron spectroscopy study of the vertical ionization energy of aqueous phenol which demonstrates the possibility of retrieving true photoelectron spectra from measurements with contributions from components with different concentration profiles.

Ultrafast excited state relaxation of a model green fluorescent protein chromophore: Femtosecond fluorescence and transient absorption study

The intrinsic excited state properties of green fluorescent protein (GFP) chromophore have inspired syntheses of its structural analogues with a motivation to replicate or improve its excited state properties for biological imaging. In this work, we report torsional-motion mediated multi-coordinate relaxation pathway to explain the excited state dynamics of a GFP chromophore analogue, (4Z)-4-(4-N,N-Dimethylaminobenzylidene)-1,2-diphenyl-1,4-dihydro-5H-imidazolin-5-one (DPI), using femtosecond fluorescence up-conversion, femtosecond transient absorption and quantum chemical calculations. The excited state dynamics of this chromophore involves the formation of an N,N-disubstituted amine twisting mediated intramolecular charge transfer state on a sub-picosecond timescale. This intramolecular charge transfer event prepares an intermediate state that undergoes a “non-volume conserving” twist motion along an exocyclic double bond. The quantum chemical computations further predict that the twist motion along the exocyclic double bond is activated and leads to the formation of a conical intersection between S1 and S0 states of DPI, which is the main nonradiative channel of deactivation. This study confirms that for a structural analogue of GFP chromophore to exhibit excited state properties like the GFP, would require preventing such nonradiative pathways, which can be achieved by restricting the twisting degree of freedom about the exocyclic double bond.

Unraveling the Mechanistic Pathway for the Dual Fluorescence in Green Fluorescent Protein (GFP) Chromophore Analogue: A Detailed Theoretical Investigation.

The photophysical properties of the para-sulfonamide (p-TsABDI) analogue of the green fluorescent protein (GFP) chromophore with both proton donating and accepting sites have been exploited in polar solvents to understand the origin of the unusual dual fluorescence nature of the chromophore. In the polar solvents, the compound undergoes structural rearrangement upon photoexcitation, leading to the ultrafast excited-state intermolecular proton transfer (ESIPT) phenomenon at the S1 surface. In this work, we employed both the static electronic structure calculations and on-the-fly molecular dynamics simulation to unravel the underlying reason for this peculiar behavior of the p-TsABDI analogue in polar solvents. To represent this adequately and provide extensive information on the ESIPT mechanism mediated by the solvent molecules, we considered explicit solvent molecules using the integral equation formalism variant of polarizable continuum (IEFPCM) model. From the static calculation analysis, we can conclude that the dual emissive behavior of the compound is ascribed to the proton transfer (PT) phenomena in the excited-state. However, based on the static calculation exclusively, it is hard to ascertain the mechanistic pathway of the PT phenomena. Hence, to investigate the dynamics and reaction mechanism for the ESIPT process, we performed the on-the-fly dynamics simulation for p-TsABDI in solvent clusters. Dynamics simulation results reveal that, based on the time lag between all the proton transfer processes, the ESIPT mechanism occurs in a stepwise manner from the benzylidene moiety of the chromophore to its imidazolinone moiety. However, the nonexistence of crossings between the S1- and S0-states confirms the PT characteristics of the reactions.

A Biomimetic Emitter Inspired from Green Fluorescent Protein.

The unique tripeptide structure of green fluorescent protein (GFP), a Ser-Tyr-Gly motif, generates the mature chromophore in situ to define the emission profiles of GFP. Here, we describe the rational design and discovery of a biomimetic fluorescent emitter, MBP, by mimicking the key structure of the Ser-Tyr-Gly motif. Through systematically tailoring the tripeptide, a family of four chromophores were engineered, while only MBP exhibited bright fluorescence in different fluid solvents with highly enhanced quantum yields. Distinct to previous hydrogen-bonding-induced fluorescence quenching of GFP chromophore analogues, the emission of MBP was only slightly decreased in protic solvents. Heteronuclear multiple bond correlation techniques demonstrated the fundamental mechanism for enhanced fluorescence emission owing to the synergy of the formation of the intramolecular hydrogen-bonding-ring structure and the self-restricted effect, which was further illustrated via theoretical calculations. This work puts forward an extraordinary approach toward highly emissive biomimicking fluorophores, which gives new insights into the emission mechanisms and photophysics of GFP-like chromophores.

Construction of a red emission fluorescent probe for selectively detection of cysteine in living cells

Cysteine (Cys) is a vital amino acid in the body, and its abnormal expression level is associated with many diseases. In this study, a novel fluorescent probe ACHB was synthesized, showing high selectivity, anti-interference ability and achieving accurate detection of cysteine. Different from most previous off–on probes, ACHB showed an on–off fluorescence response to Cys. Acrylic ester was used as a recognizer while green fluorescence protein (GFP) chromophore derivative 4-hydroxybenzylidene-imidazolinone (HBI) was used as the fluorophore. The addition of Cys leads to the hydrolysis of the red-emitting probe (613 nm), releasing a precursor with a lower fluorescent signal and showing an on–off spectral signal, which was ideal for obtaining sensitive detection with high specificity. Furthermore, the probe was successfully applied for simultaneous determination of cysteine (Cys) in living cells and biological sample (mouse serum). In conclusion, probe ACHB is a promising tool to display the intracellular cysteine concentration level, providing a good visualization method for clinical diagnosis and scientific basic research.

Synthesis and Optical Properties of the Conformationally Locked Diarylmethene Derivative of the GFP Chromophore

Synthesis and Optical Properties of the Conformationally Locked Diarylmethene Derivative of the GFP Chromophore